Membrane-type 1 matrix metalloproteinase (MT1-MMP), a transmembrane proteinase with extracellular catalytic, hemopexin-like (PEX) and hinge domains, and a short cytoplasmic tail, has been implicated in a variety of physiological and pathological processes. Unlike the other MMPs, whose genetic deficiency has minor phenotypic effects, the genetic deficiency of MT1-MMP causes severe defects in skeletal development with marked deceleration of postnatal growth, shortening of long bones, defective vascularization of the cartilage and delayed formation of secondary ossification centers. In addition, MT1- MMP-deficient mice show important defects in alveolar development and adult angiogenesis, and die by 3 weeks of age. It has been proposed that the phenotype of these mice results from the lack of the proteolytic activity of MT1-MMP. Our recently published, extensive studies have shown that MT1-MMP mediates the activation of intracellular signaling by a proteolysis-independent mechanism that stimulates cell proliferation and migration in vitro and in vivo. MT1-MMP activation of intracellular signaling is mediated by a specific sequence of the cytoplasmic tail and independent of the MT1-MMP proteolytic activity. While this novel and unexpected finding does not diminish the well-established importance of the proteolytic activity of MT1-MMP, it strongly advocates a very important role for the signaling mechanism we identified, and indicates that the phenotype of MT1-MMP null mice can result at least in part from lack of MT1-MMP-mediated signaling. The signaling mechanism we discovered is a novel, proteolysis-independent activity of MT1-MMP that may have important roles in chondrocyte proliferation in the growth plates, formation of secondary ossification centers, skeletal and alveolar development, and in adult angiogenesis. Therefore, we propose to study the physiological role of MT1-MMP- mediated intracellular signaling by developing the following Specific Aim: To generate and characterize the phenotype of a transgenic mouse with a subtle mutation that abolishes MT1-MMP-mediated intracellular signaling. We propose to generate a strain of mice expressing a mutation in the MT1-MMP cytoplasmic tail that abolishes the signaling capacity without affecting the proteolytic activity of MT1-MMP, and to compare their phenotype to those of wt and MT1-MMP null mice. We expect that expression of this mutant MT1-MMP will result in a phenotype that partially recapitulates the phenotype of MT1-MMP null mice. Therefore, the analysis of the phenotype of our mutant mice will allow us to understand the role of MT1-MMP-mediated signaling in development, normal physiology and pathology. PUBLIC HEALTH RELEVANCE: We found an unexpected, paradigm-shifting mechanism that controls cell proliferation and migration in vitro and in vivo. Therefore, we propose to generate and characterize a genetic model in mice to understand the role of this novel mechanism in development, normal physiology and in pathology. The knowledge derived from our study can provide important insight into skeletal and pulmonary alveolar development and in blood vessel formation (angiogenesis).